On tackling abuse of older people: The forensic challenges in fatal cases investigation.

The World Health Organization recently presented the priorities for tackling abuse of older people in a coordinated and strategic way. However, data on the forensic scenario is still lacking. In this context, the aim of the present work was to provide a comprehensive literature review of this inherently complex phenomenon in the post-mortem setting, in order to better characterize it from a forensic point of view. A comprehensive literature search was performed in three electronic databases following the PRISMA guidelines. Sociodemographic and medical data of victims and perpetrators, post-mortem data, types of abuse and risk factors were extracted from non-aggregated data. Forty-eight papers dealing with abuse in the post-mortem setting were included, with a predominance of case reports and case series. The review showed that neglect was the most common type of abuse and victims are predominantly older women who are abused in a domestic setting by trusted family member. To generate more and better data, expanded research in the forensic field requires standardized methods and the raise of professional awareness about abuse of older people.

Insights in opiates toxicity: impairment of human vascular mesenchymal stromal cells.

The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated ß-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths.

When the Hidden Issue of Elder Abuse Leads to Death: Do Not Neglect Elder Neglect.

ABSTRACT: It is well known that elder neglect entails a number of physical and psychological consequences, as well as being an independent risk factor for death. In the present study, we describe 5 forensic cases of death of older adults where the diagnosis of neglect only emerged postmortem through the combination of the scene investigation, the often-scarce circumstantial data, and the autopsy findings. The gathering of this information allowed the presence of risk factors-not previously assessed or reported-to be identified. Nevertheless, complete medical information was lacking both for the isolation of the victims with their caregivers and for the absence of every social and health support in self-neglect. More research is needed also in the forensic pathology field with standardized methods to contribute to prevention strategies and to avoid that victims of elder neglect remain hidden also after death.

Morphological characterization using scanning electron microscopy of fly artifacts deposited by Calliphora vomitoria (Diptera: Calliphoridae) on household materials.

Insects found at a crime scene can produce traces referred to as fly artifacts (FA) due to their movement over the corpse and the manner in which they feed upon it. These can be detrimental for carrying out criminal investigations. Confusing a FA with a genuine bloodspot can lead to misinterpretations, also taking into consideration that FA may contain a human DNA profile. The aim of the present study was to employ scanning electron microscopy (SEM) for the analysis of FA produced by Calliphora vomitoria on hard surfaces and fabrics that are commonly present at crime scenes. FA and control bloodstains were produced under experimental conditions on metal, glass, plaster, cotton, and polyester. After macroscopic analysis, FA were examined at standard low (20-40 ×), medium low (300-600 ×), and high ultrastructural (1200 ×) magnification through a SEM Stereoscan 360, Leica, Cambridge. SEM analysis enabled the identification of distinctive features of FA on hard surfaces, namely, amorphous crystals, micro-crystals with a morphology similar to those of uric or micro-crystals with a comparable morphology to cholesterol, absent in controls. Moreover, red blood cells (RBC) were absent in FA but were always present in controls. On cotton, for both FA and controls, the drop was almost completely absorbed and thus indistinguishable from the underlying fabric texture. On polyester, FA showed amorphous/crystal-like deposits and no RBC, as observed on hard surfaces, except for those showing a completely flat surface. SEM analysis appeared to be suitable for differential diagnosis between FA and genuine bloodstains on hard surfaces, although the results may be inconclusive on tested fabrics.

Assessment of the structural and functional characteristics of human mesenchymal stem cells associated with a prolonged exposure of morphine.

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs.

Integrated multidisciplinary approach in a case of occupation related planned complex suicide-peticide.

The combined event of a suicide and the killing of a pet has been hardly explored in forensic literature, but it is not rare at all. In the reported case the dead corpse of a 60-year old mentally ill woman and the carcass of a dog were found on the bed of a private apartment. In light of death scene investigation, necroscopic examination and toxicological analyses death was attributed to licit drug intoxication and self-strangulation after lethal poisoning of the dog. Due to the presence of two lethal means (cervical noose and drugs), acting in chronological order, the event was classified as a secondary complex suicide. Moreover, the woman, although mentally ill, was a psychiatrist. Thus, her professional background, namely the easy access to psychiatric drugs together with her knowledge of drug composition and properties lead to suppose an occupation related suicide. The comprehensive analysis of all the available information, including death scene investigation, occupational, necroscopic and toxicological data, resulted of the utmost importance for a proper reconstruction of the events and are recommended in complex cases such as occupation related planned complex suicides combined to the killing of pets.

Scanning electron microscopy in the identification of fly artifacts.

Bloodstain pattern analysis has a key role in crime scene reconstruction; however, it can be hampered by diverse confounding factors, such as insect activity which may lead to the production of small artifactual bloodstains, commonly referred to as fly artifacts (FA). Although several techniques aimed at distinguishing human bloodstains and FA have been developed, actually, no standardized and reproducible methodology is available. The aim of our study was to test the use of scanning electron microscopy (SEM) to distinguish human bloodstains from FA produced by Sarcophaga carnaria. FA and bloodstains have been produced on five different deposition surfaces under experimental conditions. After visual analysis, bloodstains and FA were analyzed at standard low (× 40-× 300) and high (× 600-× 1200) magnification through a Philips SEM 515. Although differential diagnosis between bloodstains and FA resulted often inconclusive at visual analysis, SEM analysis allowed the identification of additional key distinctive morphological features. In particular, on the surface of FA, small crystal-like and/or amorphous material deposits were observed. Such deposits were absent on bloodstains which, on the other hand, displayed red blood cells stacked in "rouleaux." Basing on these results and under our experimental conditions, SEM analysis resulted suitable to perform a differential diagnosis between bloodstains and FA produced from the insect activity of Sarcophaga carnaria.

Sudden unexpected death in a case of necrotizing eosinophilic myocarditis.

Acute myocarditis is related to a significant number of sudden deaths among young adults and its diagnosis is often demanded to post-mortem investigations performed on a forensic setting. Eosinophilic myocarditis (EM) is a rare form of myocarditis that is pathologically characterized by myocardial inflammation with eosinophils, often in association with elevated levels of circulating blood eosinophils. The sudden death of a 19-year-old boy with no past medical history is reported. Diagnosis of fatal acute EM was performed after a comprehensive investigation including an in-depth analysis of anamnestic and circumstantial data, and complete autopsy followed by toxicologic and cardio-pathological investigations. Discussion focuses on the forensic issues related to diagnosis and therapy of this rare form of acute myocarditis. As acute EM may be patchy, extensive myocardial sampling is mandatory in order to recognize the extent and the phase of the disease. An early diagnosis is the basis for a timely therapy, which is the key-point for prevent extensive myocardial damage, allowing a better outcome, especially when EM is acute and necrotizing. However, as demonstrated from the case herein reported, the course of EM is sometimes fulminant and does not allow any therapy nor even clinical diagnosis. Finally, this paper serves as reminder to consider this infrequent disease in differential diagnosis when facing with a sudden death, even in a young subject and in the absence of any prodrome.

Italian healthcare professionals' attitude and barriers to mandatory reporting of elder abuse: An exploratory study.

INTRODUCTION: Elder abuse in recent times has reached a remarkable international importance. This complex phenomenon is still little understood and studied in Italy. The aim of this study was to explore Italian healthcare professionals' attitude and barriers toward elder abuse, with a glimpse on the current scenario and evidences to develop an action plan. MATERIALS AND METHODS: 42 healthcare professionals filled a questionnaire with 13 open-ended questions, analyzed with a qualitative content analysis. Data were grouped under the following 4 core-themes: description of elder abuse, abuse detection and perception, barriers to reporting, and professional orientation and approach; then they were analyzed to find their central component and discussed jointly. RESULTS: The respondents described physical (64%) and psychological (50%) abuse and neglect (50%) as the three main characteristics of elder abuse; the 59% of them reported at least one experience in detecting or suspecting elder abuse. The 76% of participants believed the report has to be submitted to the Judicial Authority only in presence of compelling evidences. Furthermore, the 73% of the sample perceived that healthcare professionals are the main figures who have to cope with elder abuse, however they did not feel as their exclusive role the assessment of the patients' potential abuse. CONCLUSION: Knowledge about elder abuse was limited to physical/psychological issues and, moreover, the legal obligation to report was only poorly known. Imperative is the creation of specific education and training courses, in addition to the need for a multidisciplinary and empathetic approach.

Post-mortem thermal angiography: a pilot study on swine coronary circulation.

Thermal imaging (TI) allows the detection of thermal patterns emitted from objects as a function of their temperature in the long-infrared spectrum and produces visible images displaying temperature differences. The aim of this pilot study was to test TI to visualize the coronary circulation of swine hearts. Thirty swine hearts were prepared for ex situ coronarography, and thermal images were acquired through a FlirOne thermal camera (FLIR Systems®) paired with a Google Android Smartphone. Coronary arteries were cannulated, namely the anterior interventricular artery, the circumflex branch of the left coronary artery, and the right coronary artery. The heart was cooled, and contrast medium (CM) consisting of distilled water heated to 40 °C was injected in a coronary vessel, while thermal images were captured. These steps were repeated for each coronary vessel and under experimentally simulated coronary heart disease. Thermal imaging coronarography (TIC) allowed a clear representation of the morphology and course of the coronary vessels and of experimentally simulated coronary heart disease, moreover, demonstrated to be easy to perform during or after autopsies on ex situ hearts, non-destructive, reproducible, and cheap. On the basis of these preliminary results, TIC might allow a subsequent more focused and comprehensive cardiopathological examination of the heart, which remains mandatory for the definitive diagnosis of coronary heart disease. Although these preliminary results seem encouraging, further systematic studies on human hearts, both normal and pathological, are necessary for estimating the sensitivity and specificity of the proposed method and to draw any definitive conclusion.

Possible fatal hyperthermia involving drug abuse in a vehicle: case series.

Major interplaying causes of heat stroke (HS) and fatal hyperthermia are climate, physical activity, artificial extreme ambient temperatures, confinement in a small compartment, and effects of drugs and chemicals, combined with predispositions and complications. A common and unfortunate cause of HS is vehicular hyperthermia (VH) death. Hyperthermia in a vehicle is a type of environmental thermal disorder, involving victim's susceptibility, confinement with restraint, and possible influences of drugs and poisons, including exhaust gas containing complex fumes, carbon dioxide and monoxide. Whereas VH typically occurs when babies or young children are being left unattended in parked vehicles in direct sunlight, it has been reported only anecdotally for adult subjects. Three cases of adult fatal VH will be herein presented. In each presented case the corpse was found enclosed in a vehicle on spring/summer days. During crime scene investigation (CSI) psychoactive substances were found near to the corpses leading to the suspect of a suicidal fatal drug intoxication. Basing on this misleading suspect in Case 2 and 3 a forensic expert was not charged for the CSI and a fatal VH was not suspected nor properly investigated. Later, a comprehensive autopsy, including biochemical and toxicological analyses, excluded a death related to natural causes, fatal intoxications, ketoacidosis and traumas. On the other hand, the reconstruction of the temperature, the humidity, the heat index and the related risk of HS allowed the diagnosis of fatal VH in all the reported cases. In particular, death occurred because of the long-lasting stay into a hot parked vehicle which was facilitated from self-administration of psychoactive drugs with related neuro-depression. This case series confirms that a comprehensive CSI followed by an autopsy including histology, biochemical and toxicological analysis remains mandatory in cases of forensic interest, as well as when a corpse is found enclosed in a vehicle. Anyway, sometimes the diagnosis of heat-related fatalities remains a medley of investigative and medicolegal observations.

HIF1α protein and mRNA expression as a new marker for post mortem interval estimation in human gingival tissue.

Estimating the post mortem interval (PMI) is still a crucial step in Forensic Pathology. Although several methods are available for assessing the PMI, a precise estimation is still quite unreliable and can be inaccurate. The present study aimed to investigate the immunohistochemical distribution and mRNA expression of hypoxia inducible factor (HIF-1α) in post mortem gingival tissues to establish a correlation between the presence of HIF-1α and the time since death, with the final goal of achieving a more accurate PMI estimation. Samples of gingival tissues were obtained from 10 cadavers at different PMIs (1-3 days, 4-5 days and 8-9 days), and were processed for immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The results showed a time-dependent correlation of HIF-1α protein and its mRNA with different times since death, which suggests that HIF-1α is a potential marker for PMI estimation. The results showed a high HIF-1α protein signal that was mainly localized in the stratum basale of the oral mucosa in samples collected at a short PMI (1-3 days). It gradually decreased in samples collected at a medium PMI (4-5 days), but it was not detected in samples collected at a long PMI (8-9 days). These results are in agreement with the mRNA data. These data indicate an interesting potential utility of Forensic Anatomy-based techniques, such as immunohistochemistry, as important complementary tools to be used in forensic investigations.

A homemade furnace. Influence of occupational skills in a fire-related planned complex suicide.

The term "planned complex suicide" means the combination of more than one method of suicide, planned to prevent failure of the first method to ensure a fatal outcome. Professional skills and tools are sometimes used to plan and perpetrate the suicide. A case of planned complex suicide of a mechanical engineer working with furnaces in a tube factory is herein reported. The suicide was committed in a rudimental furnace set up in the victim's apartment using his professional skills, by assembling furniture, mattresses, books and flammable liquid present in the house. Three-dimensional models of the crime scene before and after the realization of the "homemade furnace" are proposed. The discussion will focus on the importance of a comprehensive analysis of the professional background of the victim to infer the manner of death for the identification of complex and occupation-related suicides.

The tumor microenvironment promotes cancer progression and cell migration.

The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.

HEMA but not TEGDMA induces autophagy in human gingival fibroblasts.

Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3 mmol/L of HEMA or 3 mmol/L of TEGDMA for 24, 48, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase - 3 and PARP) and autophagy (beclin - 1 and LC3B I/II) were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis.

Clostridium difficile PCR Ribotype 018, a Successful Epidemic Genotype.

Clostridium difficile infection (CDI) became a public health problem for the global spreading of the so-called hypervirulent PCR ribotypes (RTs) 027 and 078, associated with increases in the transmission and severity of the disease. However, especially in Europe, several RTs are prevalent, and the concept of hypervirulence is currently debated. We investigated the toxin and resistance profiles and the genetic relatedness of 312 C. difficile strains isolated in a large Italian teaching hospital during a 5-year period. We evaluated the role of CDI-related antibiotic consumption and infection control practices on the RT predominance in association with their molecular features and transmission capacity. Excluding secondary cases due to nosocomial transmission, RT018 was the predominant genotype (42.4%) followed by RT078 (13.6%), while RT027 accounted for 0.8% of the strains. RT078 was most frequently isolated from patients in intensive care units. Its prevalence significantly increased over time, but its transmission capacity was very low. In contrast, RT018 was highly transmissible and accounted for 95.7% of the secondary cases. Patients with the RT018 genotype were significantly older than those with RT078 and other RTs, indicating an association between epidemic RT and age. We provide here the first epidemiological evidence to consider RT018 as a successful epidemic genotype that deserves more attention in clinical practice.

Epidemic MRSA clone ST22-IV is more resistant to multiple host- and environment-related stresses compared with ST228-I.

BACKGROUND: ST22-IV is a successful hospital-associated MRSA clone. Due to its known ability to replace other MRSA clones in hospitals, it became a dominant clone in Europe and beyond. So far, there are no studies investigating the relationship between the epidemiological success of MRSA clones and their capacity to withstand commonly encountered stresses. METHODS: We investigated the fitness of ST22-IV in comparison with the replaced clone ST228-I, evaluating its resistance to oxidative stress, autolytic activity, growth at high osmolarity and in acid and alkaline environments and survival under desiccation and heat shock. We also compared their phenotypic characteristics and examined the impact of antibiotic consumption on epidemiological success. RESULTS: Here we demonstrate that the dominance of ST22-IV is linked neither to changes in antibiotic consumption nor to acquisition of additional resistances over time. Strong α-haemolysin activity, the production of ß-haemolysin and the presence of an active agr could partly explain the virulence of ST22-IV previously observed in a murine model of pneumonia. Most importantly, we show that ST22-IV compared with ST228-I, besides retaining susceptibility to most antibiotics over time, has a superior capacity to survive under all stress conditions tested, which bacteria commonly face during their life cycle. CONCLUSIONS: Our results support our hypothesis that ST22-IV has a fitness advantage over ST228-I. This fitness advantage could have allowed ST22-IV to displace ST228-I without acquiring additional resistances and could help explain its epidemic success in hospital settings and its spread in Europe and beyond.

Elder abuse awareness in Italy: analysis of reports to the Prosecutor Office of Bologna.

BACKGROUND: In Italy, the prevalence of elder abuse is estimated from 10 to 12.7 %. Even if the elderly are not specifically protected by the Italian Penal Code, it contains several articles punishing crimes against disadvantaged groups in which elders may be included. Article 572 about mistreatment of family members or cohabitants, and article 643 about circumvention of incapable best fit elder abuse or neglect and financial fraud and exploitation, respectively. AIMS: To evaluate the prevalence of reported elder abuse in Bologna and province (Italy) compared to available data on prevalence obtained by population-based surveys. METHODS: Reports to the Prosecutor Office about articles 572 and 643 in the period from 2000 to 2013 were collected and analyzed according to victim's age at the report time. RESULTS: For article 572 a total of 3,713 reports were found. The prevalence of reports ranged from 0.005 to 0.024 % for victims older than 65 years. Concerning article 643 a total of 786 reports were detected, with a prevalence ranging from 0.007 to 0.016 % for victims older than 65 years. DISCUSSION AND CONCLUSION: A large gap was found between reported crimes related to elder abuse and the estimated prevalence of elder abuse in Italy. Our results showed that in Italy efforts to improve strategic approach on elder abuse as well as research at various levels following the examples of other abusive situations are needed in both health and legal systems.

Simulating tumor microenvironment: changes in protein expression in an in vitro co-culture system.

BACKGROUND: The role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics. The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. METHODS: The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24 h to 7 days. Cell morphology and organization were studied using phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloprotease 1 (MMP1) was investigated by Real Time PCR and Western Blotting. RESULTS: The results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. CONCLUSIONS: We concluded that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity.

Ultrastructural changes in human gingival fibroblasts after exposure to 2-hydroxy-ethyl methacrylate.

Polymerized resin-based materials are successfully utilized in medical applications. One draw- back is the release of monomers from the matrix due to an incomplete polymerization or degradation processes. Released monomers can diffuse in the systemic circulation and induceadverse effects to biological tissues. Although there are many hypotheses about the induction of cell death by resin monomers, the underlying mechanisms are still under discussion. The aim of the study was to investigate the morphological modifications in human gingival fibroblasts exposed to 2-hydroxy-ethyl methacrylate (HEMA) to better elucidate the mechanism of cell death induced by resin monomers. Primary cultures of gingival fibroblasts were exposed to 3mM HEMA for 24 h, 72 h, 96 h. Morphological investigations were performed by scanning and transmission electron microscopy, while western blot for caspase-3 was carried out to ver- ify apoptosis. Electron microscopy images showed deep changes in the cell surface and cyto- plasm after 72 h and 96 h of HEMA treatment. Autophagic vesicles were easily observed just after 24 h. Cleaved caspase-3 was detected after 72 h of treatment. These findings suggest that resin based materials induced cell death by the cooperation of apoptosis and autophagy mecha- nisms. The understanding of these mechanisms will lead to the development of smart biomate- rials without or with low adverse effects.